P2&#39;-modified hydroxamic acid collagenase inhibitors

ABSTRACT

Compounds of general formula I: ##STR1## Wherein: R 1 , R 2 , R 3 , R 4 , R 5 , and R 6  are variables. These compounds have collagenase inhibition activity and are useful in the management of disease involving collagen degradation. Its uses include rheumatoide arthritis, corneal ulceration, osteoporosis, periodontitis, gingivitis and tumor invasion.

This is a divisional of application Ser. No. 07/760,741, filed Sep. 16,1991, now U.S. Pat. No. 5,300,674, patented Apr. 5, 1994.

A number of small peptide like compounds which inhibit metalloproteinasehave been described. Perhaps the most notable of these are thoserelating to the angiotensin converting enzyme (ACE) where such agentsact to blockade the conversion of the decapeptide angiotensin I toangiotensin II, a potent pressor substance. Compounds of this type aredescribed in EP-A-0012401.

Certain hydroxamic acids have been suggested as collagenase inhibitor asin U.S. Pat. No. 4,599,361, WO-A-9005716 and WO-A-9005719. Otherhydroxamic acids have been prepared as ACE inhibitors, for example, inU.S. Pat. No. 4,105,789, while still others have been described asenkephalinase inhibitors as in U.S. Pat. No. 4,495,540.

The hydroxamic acids of the current invention act as inhibitors ofmammalian collagenase which initiates collagen breakdown. There isevidence implicating collagenase as one of the key enzymes in thebreakdown of articular cartilage and bone in rheumatoid arthritis(Arthritis and Rheumatism, 20, 1231-1239, (1977)). Potent inhibitors ofcollagenase are useful in the treatment of rheumatoid arthritis andrelated diseases in which collagenolytic activity is important. Thesediseases include corneal ulceration, osteoporosis, periodontitis,gingivitis and tumour invasion.

The current invention relates to a series of hydroxamic acids, offormula I, which act as inhibitors of metalloproteinase, theirpreparation, pharmaceutical compositions containing them, and theintermediates involved in their preparation. ##STR2## Wherein: R¹ ishydrogen, C₁ -C₆ alkyl, phenyl, substituted phenyl, phenyl (C₁ -C₆alkyl), or heterocyclyl; or R¹ is ASO_(n) R⁷

wherein A represents a C₁ -C₆ hydrocarbon chain, optionally substitutedwith one or more C₁ -C₆ alkyl, phenyl or substituted phenyl groups

n=0,1,2;

R⁷ is C₁ -C₆ alkyl, phenyl, substituted phenyl, phenyl (C₁ -C₆ alkyl),heterocyclyl, (C₁ -C₆ alkyl)acyl, thienyl or phenacyl;

R² is hydrogen, C₁ -C₆ alkyl, C₂ -C₆ alkenyl, phenyl (C₁ -C₆ alkyl) orcycloalkyl(C₁ -C₆ alkyl);

R³ and R⁴ are selected from hydrogen, halogen, cyano amino, amino(C₁-C₆)alkyl, amino di(C₁ -C₆)alkyl, amino(C₁ -C₆)alkylacyl, aminophenacyl,amino (substituted) phenacyl, amino acid or derivative thereof, hydroxy,oxy(C₁ -C₆)alkyl, oxyacyl, formyl, carboxylic acid, carboxamide,carboxy(C₁ -C₆) alkylamide, carboxyphenylamide, carboxy(C₁ -C₆) alkyl,hydroxy(C₁ -C₆)alkyl, (C₁ -C₆)alkyloxy(C₁ -C₆) alkyl or acyloxy(C₁-C₆)alkyl, (C₁ -C₆)alkylcarboxylic acid, or (C₁ -C₆) alkylcarboxy(C₁-C₆) alkyl; or

R³ is OCH₂ COR⁸ and R⁴ is hydrogen;

wherein R⁸ is hydroxyl, C₁ -C₆ oxyalkyl, C₁ -C₆ oxyalkylphenyl, amino,C₁ -C₆ aminoalkyl, C₁ -C₆ aminodialkyl, C₁ -C₆ aminoalkylphenyl, anamino acid or derivative thereof; or

R³ is OCH₂ CH₂ OR⁹ and R⁴ is hydrogen;

wherein R⁹ is C₁ -C₆ alkyl, C₁ -C₆ alkylphenyl, phenyl, substitutedphenyl, (C₁ -C₆ alkyl) acyl, or phenacyl; or

R³ is OCH₂ CN and R⁴ is hydrogen

R⁵ is hydrogen or C₁ -C₆ alkyl, or (C₁ -C₆) alkylphenyl;

R⁶ is hydrogen or methyl; or a salt thereof;

specifically excluded are compounds wherein:

R³ =R⁴ =hydrogen or

R³ =R⁴ =hydroxy or

R³ =hydrogen and R⁴ =oxybenzyl or

R³ =hydrogen and R⁴ =oxy(C₁ -C₆ alkyl).

Hereafter in this specification the term "compound" includes salt unlessthe context requires otherwise.

As used herein the term "C₁ -C₆ alkyl" refers to a straight or branchedchain alkyl moiety having from one to six carbon atoms, including forexample, methyl, ethyl, propyl, isopropyl, butyl, t-butyl, pentyl, hexyland the like.

The term "C₂ -C₆ alkenyl" refers to a straight or branched chain alkylmoiety having two to six carbons and having in addition one double bond,of either E or Z stereochemistry where applicable. This term wouldinclude, for example, vinyl, 1-propenyl, 1- and 2-butenyl,2-methyl-2-propenyl etc.

The term "cycloalkyl" refers to a saturated alicyclic moiety having from3 to 8 carbon atoms and includes for example, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl and the like.

The term `heterocyclyl` refers to a saturated or unsaturated ringcontaining at least one hereto atom such as nitrogen, oxygen or sulphurand includes for example, furan, pyrrole, thiophene, morpholine,pyridine, dioxane, imidazoline, pyrimidine and pyridazine.

The term "substituted", as applied to a phenyl or other aromatic ring,means substituted with up to four substituents each of whichindependently may be C₁ -C₆ alkyl, C₁ -C₆ alkoxy, hydroxy, thiol, C₁ -C₆alkylthiol, amino, halo (including fluoro, chloro, bromo and iodo),trifluoromethyl, nitro, --COOH, --COONH₂ or --CONHR^(A), wherein R^(A)represents a C₁ -C₆ alkyl group or the characteristic side chain of anamino acid such as alanine, valine, leucine, isoleucine, phenylalanine,tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine,asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine orhistidine.

The term "amino acid"means one of the following R or S amino acids:glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine,tryptophan, serine, threonine, cysteine, methionine, asparagine,glutamine, lysine, histidine, arginine, glutamic acid and aspartic acid.

Derivatives of amino acids include acid halides, esters and substitutedor unsubstituted amides, for example N methyl amide.

There are several chiral centres in the compounds according to theinvention because of the presence of asymmetric carbon atoms.

The presence of several asymmetric carbon atoms gives rise to a numberOf diastereomers with the appropriate R or S stereochemistry at eachchiral centre. The invention is understood to include all suchdiastereomers and mixtures thereof.

Preferred compounds include those in which, independently or incombination:

R¹ represents a hydrogen atom or a C₁ -C₄ alkyl, or phenyl group; or

R¹ represents ASO_(n) R⁷ in which A is C₁ -C₄ hydrocarbon chain alkyl(for example methylene), n=0, and R⁷ is a phenyl, substituted phenyl orthienyl group;

R² represents a C₁ -C₅ alkyl (for example isobutyl) group;

R³ represents cyano, aminoalkylacyl or OCH₂ COR⁸ and R⁴ is hydrogen;

Wherein R⁸ represents a hydroxyl group, C₁ -C₆ oxyalkyl, amino, C₁ -C₆aminoalkylphenyl, C₁ -C₆ aminodialkyl, or an amino acid or derivativethereof;

R.sup.≡ represents a C₁ -C₄ alkyl (for example methyl) group;

R⁶ represents a hydrogen atom.

Particularly preferred compounds include:

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxlicacid)phenylalanine-N-methylamide;

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxy-N-methylamide)phenylalanine-N-methylamide;

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4oxymethylcarboxy-beta-alanine)phenylalanine-N-methylamide;

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4-(oxymethylcarboxy-glycine)phenylalanine-N-methylamide;

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4oxymethylcarboxy-N-benzylamide)phenylalanine-N-methylamide;

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4-cyano)phenylalanine-N-methylamide;

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4-acetamido)phenylalanine-N-methylamide.

Compounds of the general formula I may be prepared by any suitablemethod known in the art and/or by the following process, which itselfforms part of the invention.

According to a second aspect of the invention, there is provided aprocess for preparing a compound of general formula I as defined above,the process comprising:

(a) deprotecting (for example by hydrogenation) a compound of generalformula II ##STR3## Wherein: R¹, R², R³, R⁴, R⁵ and R⁶ are as defined inthe general formula I and Z represents a suitable protective group (e.g.tert-butyl, tertbutylsilyl, benzyl or substituted benzyl);

(b) reacting a compound of general formula III ##STR4## Wherein: R¹, R²,R³, R⁴, R⁵, and R⁶ are as defined in the general formula I, withhydroxylamine or a salt thereof; and

(c) optionally after step (a) or step (b) converting a compound ofgeneral formula I into another compound of general formula I.

Compounds of general formula I which are sulphoxide or sulphones can bederived from thio compounds of general formula I by oxidation.Alternatively, compounds of general formula II, or III which containsulphur can be oxidised.

A compound of general formula II can be obtained by coupling, forexample by conventional coupling techniques, a compound of generalformula III with an O-protected hydroxylamine of formula NH₂ OZ; whereinZ is as defined in general formula II.

A compound of general formula III can be prepared by

(a) de-esterifying (for example under acid or base catalysis) a compoundof general formula IV ##STR5## Wherein: R¹, R², R³, R⁴, R⁵, and R⁶ areas defined in the general formula I, and R¹⁰ represents a C₁ -C₆ alkylor benzyl group; or

(b) by reacting a compound of general formula V ##STR6## Wherein: R²,R³, R⁴, R⁵, and R⁶ are as defined in the general formula I,

either with a thiol of general formula R⁷ SH, wherein R⁷ is as definedin general formula I, to give a compound of general formula III in whichR¹ is ASO_(n) R⁷, A represents a methylene group and n is 0.

or with compound R¹ X where R¹ is benzyl or substituted benzyl and X isF, Cl, Br of I in the presence of a palladium catalyst to provide acompound of general formula VI ##STR7## Wherein: R², R³, R⁴, R⁵, and R⁶are as defined in the general formula I, and R¹ is benzyl or substitutedbenzyl, which may be converted to a compound of general formula IIIwherein R² is benzyl or substituted benzyl, by hydrogenation; or

(c) by converting a compound of general formula III into anothercompound of general formula III.

A compound of general formula V can be prepared from a compound ofgeneral formula IV ##STR8## Wherein: R², R³, R⁴, R⁵, and R⁶ are asdefined in the general formula I, R¹ is carboxybenzyl or carboxy (C₁-C₆) alkyl and R10 is benzyl or (C₁ -C₆) alkyl, and R¹⁰ is benzyl or (C₁-C₆) alkyl,

by de-esterification (for example by hydrogenation) followed by reactionwith formaldehyde in the presence of morpholine.

A compound of general formula IV can be prepared

(a) By reacting, for example by conventional coupling techniques, anacid of formula VII, or an activated ester derivative thereof, ##STR9##Wherein: R¹ and R² are as defined in the general formula I, and R¹⁰ isas defined above.

with an amine of general formula VIII ##STR10## Wherein: R³, R⁴, R⁵, andR⁶ are as defined in the general formula I;

(b) by converting a compound of general formula IV into another compoundof general formula IV.

An amine of general formula VIII can be prepared by deprotection (forexample with trifluoroacetic acid) of a compound of general formula IX##STR11## Wherein: R¹² is a conventional amine protecting group and R³,R⁴, R⁵ and R⁶ , are as defined in general formula I.

A compound of general formula IX may be prepared by coupling an acid ofgeneral formula X ##STR12## Wherein: R³ and R⁴, are defined as ingeneral formula I, with an amine of general formula XI ##STR13##Wherein: R⁵ and R⁶ are as defined in general formula I

A compound of general formula X may be prepared

(a) by reaction of an aryl halide of general formula XII ##STR14##Wherein R3 and R4 are as defined in general formula I with a glycinateanion equivalent of formula XIII ##STR15## followed by acid hydrolysis,protection of the amino function and base catalysed release of thecarboxylic acid; or

(b) by converting a compound of general formula X to another compound ofgeneral formula X.

A compound of general formula VII may be prepared by reaction of acompound of general formula XIV ##STR16## Wherein: R² is as defined inthe general formula I, R¹ is hydrogen, R¹⁰ is as described above and R¹³is a chiral auxiliary for example as described by Evans (J. Amer. Chem.Soc., 104, 1737, (1982)).

with lithium hydroxide/hydrogen peroxide.

A compound of general formula XIV may be produced by alkylation of theanion of a compound of general formula XV ##STR17## Wherein: R² is asdefined in the general formula I and R¹³ is a chiral auxiliary,

with an alkylating agent of general formula XVI. ##STR18## Wherein: R¹⁰is as described above and X is a leaving group, for example bromide,iodide or triflate.

Compounds of general formulae XI, XII, XIII, XV and XVI and otherreagents are either available commercially or can be synthesised bysimple chemical procedures.

The potency of compounds of the present invention to act as inhibitorsof collagenase was determined by the procedure of Cawston and Barrett,(Anal. Biochem., 99, 340 -345, 1979) whereby a 1 mM solution of theinhibitor being tested or dilutions thereof is incubated at 37° C. for16 hours with collagen and collagenase (buffered with Tris HCl --CaCl₂ ;pH 7.6). The collagen is acetylated ¹⁴ C collagen prepared by the methodof Cawston and Murphy (Methods in Enzymology, 80, 711, (1981)). Thesamples are centrifuged to sediment undigested collagen and an aliquotof the radioactive supernatant removed for assay on scintillationcounter as a measure of hydrolysis. The collagenase activity in thepresence of 1 mM inhibitor, or a dilution thereof, is compared toactivity in a control devoid of inhibitor and the results reported asthat inhibitor concentration effecting 50% inhibition of thecollagenase.

In a further aspect of the invention there is provided the use of acompound of general formula I in medicine, particularly in a method oftreatment of diseases in which collagenolytic activity is important.

In another aspect of the invention there is provided the use of acompound of general formula I in the preparation of an agent for thetreatment of diseases in which collagenolytic activity is important.

The invention also provides a pharmaceutical composition comprising oneor more compounds of general formula I in association with one or morenon-toxic pharmaceutically acceptable carriers and/or diluents and/oradjuvants. Other active ingredients may also be included in thecompositions of the invention.

The compositions of the present invention may be formulated foradministration by any route depending on the disease being treated. Thecompositions may be in the form of tablets, capsules, powders, granules,lozenges, liquid or gel preparations, such as oral, topical, or sterileparental solutions or suspensions.

Tablets and capsules for oral administration may be in unit dosepresentation form, and may contain conventional excipients. Examples ofthese are bindings agents such as syrup, acacia, gelatin, sorbitol,tragacanth, and polyvinylpyrollidone; fillers for example lactose,sugar, maize-starch, calcium phosphate, sorbitol or glycine; tablettinglubricants, for example magnesium sterate, talc, polyethylene glycol orsilica; disintegrants, for example potato starch, or acceptable wettingagents such as sodium lauryl sulphate. The tablets may be coatedaccording to methods well known in normal pharmaceutical practice. Oralliquid preparations may be in the form of, for example, aqueous or oilysuspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives such as suspending agents, for example sorbitol,syrup, methyl cellulose, glucose syrup,gelatin, hydrogenated ediblefats; emulsifying agents, for example lecithin, sorbitan monooleate, oracacia; non-aqueous vehicles (which may include edible oils), forexample almond oil, fractionated coconut oil, oily esters such asglycerine, propylene glycol, or ethyl alcohol; preservatives, forexample methyl or propyl p-hydroxybenzoate or sorbic acid, and ifdesired conventional flavouring or colouring agents.

The dosage unit involved in oral administration may contain from about 1to 250 mg, preferably from about 25 to 250 mg. A suitable daily dose fora mammal amy vary widely depending on the condition of the patient.However, a dose of about 0.1 to 300 mg/kg body weight, particularly fromabout 1 to 100 mg/kg body weight may be appropriate.

For topical application to the skin the drug may be made up into acream, lotion or ointment. Cream or ointment formulations that may beused for the drug are conventional formulations well known in the art,for example, as described in standard text books of pharmaceutics suchas the British Pharmacopoeia.

For topical applications to the eye, the drug may be made up into asolution or suspension in a suitable sterile aqueous or non-aqueousvehicle. Additives, for instance buffers such as sodium metabisulphiteor disodium edeate; preservatives including bactericidal and fungicidalagents, such as phenyl mercuric acetate or nitrate, benzalkoniumchloride or chlorohexidine, and thickening agents such as hypromellosemay also be included.

The dosage employed for the topical administration will, of course,depend on the size of the area being treated. For the eyes each dosewill be typically in the range from 10 to 100 mg of the drug.

The active ingredient may also be administered parenterally in a sterilemedium. The drug depending on the vehicle and concentration used, caneither be suspended or dissolved in the vehicle. Advantageously,adjuvants such as local anaesthetic, preservatives and buffering agentscan be dissolved in the vehicle.

For use in the treatment of rheumatoid arthritis the compounds of thisinvention can be administered by the oral route or by injectionintra-articularly into the affected joint, The daily dosage for a 70 kgmammal will be in the range of 10 mgs to 1 gram.

The following examples illustrate the invention, but are not intended tolimit the scope in any way.

The following abbreviations have been used in the Examples:

DCM--Dichloromethane

DMF--N,N-Dimethylformamide

HOBT--Hydroxybenztriazole

NMM--N-Methylmorpholine

TFA--Trifluoroacetic acid

THF--Tetrahydrofuran

WSCDI--N-(Dimethylaminoethyl)-N'-ethylcarbodiimide.

EXAMPLES Example 1

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxylicacid)-phenylalanine-N-methylamide

Example 1a

N-(4-Methylpentanoyl)-4S-phenylmethyl-2-oxazolidinone

A dry 500 ml flask equipped with a magnetic stirrer was charged with(S)-4-Phenylmethyl-2-oxazolidinone (17.72 g, 0.1 mol), this was cappedWith a rubber septum and flushed with N₂. Anhydrous THF (300 ml) wasadded via cannula and the resulting solution was cooled to -78° C. in anacetone/dry ice bath. A solution of 1.47M n-butyllithium in hexane (68.4ml, 0.101 mol) was transferred via cannula to a dry, septum-stoppered100 ml dropping funnel. This was added dropwise to the THF solution over10 minutes.

4-methyl valeric acid chloride (14.80 g 0.11 mol) was added in oneportion by syringe after completion of the addition of n-butyllithium.The resulting solution was stirred at -78° C. for 30 minutes and thenallowed to warm to ambient temperature over 30 minutes. Excess of theacid chloride was quenched by the addition of aq. NH₄ Cl (60 ml) and thebulk-of the solvent was removed. The resulting slurry was extracted withdichloromethane (2×80 ml). The combined organic extracts were washedwith 1M NaOH (75 ml), brine (75 ml), dried (Na₂ SO₄ anhyd.) andfiltered. The solvent was removed to yield a yellow oil (29.20 g, 106%).

Analysis calculated for C₁₆ H₂₁ NO₃ MWt=275.34 delta_(H) (250 MHz,CDCl₃), 0.97 (6H, d, C(CH₃)2, J=6.2 Hz), 1.53-1.76 (3H, m, CH₂ CHMe₂),2.78 (1H, dd, CH₂ Ph, J=9.5 Hz), 2.85-3.05 (2H, m, COCH₂) 3.30 (1H, dd,CH₂ Ph, J=3.3 Hz) 4.16-4.25 (2H, m, CH₂ OCO) 4.63-4.73 (1H, m, CHBnz)7.19-7.34 (5H, m, C₆ H₅)

Example 1b

N(4-(t-Butoxy)-2R-isobutylsuccinyl)-4S-phenylmethyl-2oxazolidinone

N-(4-Methylpentanoyl)-4S-phenylmethyl-2-oxazolidinone (20 g, 0.0726 mol)was placed in a dry 1 liter 3-necked flask to which was added dry THF(400 ml). The mixture was kept under a stream of Argon and cooled to-78° C. (dry ice/acetone). Sodium hexamethyldisilylamide (1M solution inTHF, 0.0726 mol, 72.6 ml) was added dropwise through a dropping funnel(it was added to the funnel via syringe). After stirring for 20 minutes,t-butylbromoacetate (21.02 g, 15.8 ml, 0.1089 mol, 1.5 equiv.) was addeddropwise over 1 minute, to give an orange solution. The mixture was keptat -78° C. and allowed to warm to -50° C. over 2 hours (after which timeit turned pink). The reaction was then quenched by adding acetic acid(10.90 g, 10.4 ml, 0.1815 mol, 2.5 equiv.) in ether (50 ml) at -50° C.whereupon the solution became colourless. The solvent was removed andthe resulting slurry partitioned between ethyl acetate and brine. Theethyl acetate layer was washed once with brine and the original brinelayer was back-extracted with ethyl acetate. The combined organic layerswere dried and the solvent removed, giving a yellow oil whichcrystallised on cooling overnight to yield the title compound as acrystalline solid (21.36 g, 76%).

Analysis calculated for C₂₂ H₃₁ O₅ N MWt=389.48 delta_(H) (250 MHz,CDCl₃) 0.91-0.96 (6H, dd, CMe₂, J=4.5 Hz), 1.44 (9H, s, CMe₃) 1.24-1.72(3H, m, CH₂ CHMe₂), 2.49 (1H, dd, CH₂ Ph, J=4.6 Hz), 2.72 (1H, dd, CH₂CO₂ CH(CH₃)3, J=2.3 Hz), 3.36 (1H, dd, CH₂ Ph, J=3.25 Hz), 4.16-4.18(2H, m, CH₂ OCO), 4.20-4.35 (1H, m, CH--CO), 4.62-4.72 (1H, m, CHBz),7.24-7.38 (5H, m, C₆ H₅) [alpha]²⁵ D=+66.9 (c=1, MeOH)

Example 1c

4-(t-Butoxy)-2R-isobutylsuccinic acid

N(4-(t-Butoxy)-2R-isobutylsuccinyl)-4S-phenylmethyl-2-- oxazolidinone(15.30 g, 0.039 mol) was placed in a 1 liter flask with a stirrer barand to it was added 750 ml of 4.1 THF:H₂ O. This solution was stirredand cooled to 0° C. (ice/acetone bath) then 60% aq. H₂ O₂ (4.5 ml, 0.157mol, 4 equiv) was added via syringe over 5 mins, followed by Li(OH)₂(2.65 g, 0.063 mol, 1.6 equiv.) in 100 ml water. The reaction mixturewas stirred for 1h at 0° C. TLC (10% methanol/dichloromethane) showedcomplete reaction (product gave a yellow spot on TLC on staining withbromocresol green and heating). The reaction mixture was quenched withNaNO₂ (10.88 g, 0.157 mol, 4 equiv.), the final pH was 12-13. THF wasremoved in-vacuo and the aqueous layer extracted with dichloromethane(3×200 ml) to recover the chiral auxliary. The organic extracts weredried (MgSO₄ anhyd.), solvent removed in-vacuo and the resulting solidchiral auxiliary (7.05 g, 0,039 mol, 100%) recrystallised from ethylacetate/hexane (2:1)

[alpha]²⁵ D=13.0° (x=1, MeOH) [alpha ]²⁵ D 4.9° (c=1, EtOH)

The aqueous layer was cooled in an ice bath and acidified to pH 5-6 with2M HCl. The resulting cloudy solution was extracted with ethyl acetate(4×200 ml), readjusting the pH to 5-6 in between extraction. Thecombined organic extracts were dried over MgSO₄, filtered and thesolvent was removed to yield the title compound as a pale yellow oil(8.21 g, 91%).

delta_(H) (250 MHz, CDCl₃) 0.93 (6H, dd, J=7, 8 Hz), 1.28 (1H, m), 1.64(1H, m), 2.38 (1H, dd, J=16, 5 Hz), 2.59 (1H, dd, J=16, 9 Hz), 2.85 (1H,m). [alpha]²⁵ D=+10.4 (c=1, MeOH)

Example 1d

Pentafluorophenyl-4-(t-butoxy)-2R-isobutylsuccinate

A solution of the chiral acid (from example 1c, 5.0 g, 21.7 mmol) andpentafluorophenol (8.0 g, 43 mmol) in dichloromethane (50 ml) was cooledto 0° C. before dropwise addition of N-methylmorpholine (2.7 g, 26.7mmol) followed by water-soluble carbodiimide (5.5 g, 28.7 mmol) inseveral portions. After the WSCDI had dissolved, a small amount of whiteinsoluble material remained which did not dissolve on addition ofN,N-dimethylformamide (5 ml). The mixture was allowed to warm to roomtemperature and was then stirred overnight at room temperature.

Solvents were removed on a rotary evaporator and the residue wasresuspended in dichloromethane (100 ml) and washed successively with 1MHCl (2×200 ml), 0.5M Na₂ CO3 (2×200 ml) and brine (200 ml) and dried(Na₂ SO₄). TLC (CH₂ Cl₂) showed a single UV-active spot (R_(f) ca.0.8)with a small amount of brown baseline impurity. The solution wastherefore evaporated to a brown oil-and flushed through a silica column(2×20 cm) with dichloromethane. UV-positive fractions were pooled andevaporated to give the pentafluorophenol ester as a pale yellow oil(8.31 g, 97%).

C₁₈ H₂₁ F₅ O₄ MWt=396.35 i.r. (neat) 1785, 1732 cm⁻¹ delta_(H) (250 MHz,CDCl₃) 3.23 (1H, m), 2.74, 2.52 (2H, ddd, J=9.3, 5.2, 16.8 Hz), 1.75(2H, m), 1.46 (10H, s and m), 0.98, 0.96 (6H, 2d, J=6.6 Hz) delta_(c)(250 MHz, CDCl₃) 171.3, 170.3, 143.2-138.9, 81.4, 41.0, 39.5, 37.7,28.0, 25.8, 22.6, 22.1

Example 1e

O-Benzyl-L-tyrosine N-methylamide

N-Boc-O-benzyl tyrosine methylamide (5.29 g, 13.8 mmol) was taken up inCH₂ Cl₂ (100 ml). To the solution at 0° C. TFA (10 ml) was addeddropwise and the solution allowed to warm to ambient temperature. After4 hours the solvent and TFA were removed under vacuum. Any remaining TFAwas quenched with saturated NaHCO₃ solution (100 ml). The reactionmixture was extracted using CH₂ Cl₂ (100 ml) and washed with saturatedNaHCO₃ solution (100 ml) and brine (100 ml). The CH₂ Cl₂ layer was driedover Na₂ SO₄ and the solvent was removed under vacuum to give a whitesolid, which was recrystallised from ethyl acetate/hexane to yield thetitle compound as a white crystalline solid (3.35 g, 85.4%).

delta_(H) (250 MHz, CDCl₃) 7.45-7.33 (5H, bm, Bn-H), 7.23 (1H, bs,CONHMe), 7.13 (2H, d, J=8.5 Hz, Ar-H), 6.93 (2H, d, J=8.6 Hz, Ar-H),5.06 (2H, s, CH₂), 3.56 (1H, dd, J=5.1, 4.0 Hz, CH), 3.20 (1H, dd,J=9.8, 4.0 Hz, H of CH₂), 2.82 (3H, d, J=5.0 Hz, NHCH₃), 2.65 (1H, dd,J=9.3, 4.5 Hz, H of CH₂), 1.35 (2H, bs, NH₂).

Example 1f

[4-(t-Butoxy)-2R-isobutylsuccinyl]-L-4-benzyloxy)phenylalanine-N-methylamide

To a stirred solution of the 0-benzyl-L-tyrosine-N-methylamide (fromexample 1e, 3 g, 10.6 mmol) in DMF (100 ml) was added the chiralpentafluorophenyl ester (from example 1d, 8.37 g, 21.1 mmol). Theresulting solution was stirred at room temperature overnight. The DMFwas removed under vacuum. The residue was taken up in CH₂ Cl₂ (200 ml)and washed with saturated NaHCO₃ (2×100 ml), citric acid (2×100 ml) andbrine (100 ml). The organic layer was dried over MgSO₄ and the solventremoved under vacuum to give a clear oil. Flash chromatography (flashsilica, CH₂ Cl₂ to 5% MeOH/CH2Cl12) gave the title compound as a paleyellow solid (4.95 g, 94%).

C₂₉ H₄₀ N₂ O₅ MWt=496.65 delta_(H) (250 MHz, CDCl₃) 7.45-7.31 (5H, m,CH-21 to 25), 7.15 (2H, d, J=8.6 Hz, CH-9,11), 6.91 (2H, d, J=8.6 Hz,CH-8,12), 6.30 (1H, d, J=7.8 Hz, CONH), 5.92 (1H, m, CONHMe), 5.04 (2H,s, CH₂₋₁₉), 4.50 (1H, q, J=7.8 Hz, CH-5), 3.0 (1H, dd, J=6.3, 6.2 Hz,CH₂ -6a), 2.82 (1H, dd, J=7.8 Hz, CH₂ -6b), 2.70 (3H, d, J=4.8 Hz, CH₃-13), 2.61 (1H, m, CH-3), 2.46 (2H, m, CH₂ -2a, 2b), 1.52 (2H, m, 1.44,CH₂ -15), (9H, s, CH₃ -27, 28, 29), 1.19 (1H, m, CH-16), 0.88, 0.85 (6H,2d, J=6.5, 6.3 Hz, CH₃ -17, 18).

Example 1g

[4-(t-Butoxy)-2R-isobutylsuccinyl]-L-(4-hydroxy)phenylalanine-N-methylamide

[4-(t-Butoxy)-2R-isobutylsuccinyl)-L-(4-benzyloxy)phenylalanine-N-methylamide (2.19 g, 4.4 mmol) was taken up in 10%cyclohexene/ethanol (30 ml) and 10% Pd/charcoal (0.219 g) added. Themixture was then heated under reflux. After 3 hours the hot solution wasfiltered through glass fibre paper and the black solid washed withmethanol. The filtrate was concentrated under reduced pressure to givethe title compound as a white foam (1.78 g, 99%).

C₂₂ H₃₄ N₂ O₅ MWt=405.6 delta_(H) (250 MHz, CDCl₃) 7.08 (2H, d, J=8.6Hz, CH-9,11), 6.76 (2H, d, J=8.6 Hz, CH-8, 12), 6.35 (1H, d, J=8.0 Hz,CONH), 5.91 (1H, m, CONHMe), 4.50 (1H, q, J=7.9 Hz, CH-5), 3.06 (1H, dd,J=6.2 Hz, CH₂ -6a), 2.96 (1H, dd, J=7.9 Hz, CH₂ -6b), 2.71 (3H, d, J=4.8Hz, CH₃ -13), 2.61 (1H, m, CH-3), 2.48 (2H, m, CH₂ -2a, 2b), 1.52 (2H,m, CH₂ -15), 1.44 (9H, s, CH₃ -20, 21, 22), 1.25 (1H, m, CH-16), 0.86(6H, d, J=6.4 Hz, CH₃ -17, 18). delta_(C) (250 MHz, CDCl₃) 173.8, 170.4,154.2, 128.8, 126.3, 114.2, 79.8, 76.1-75.1, 53.7, 39.6, 36.7, 36.1,26.6, 24.8, 24.2, 21.2, 20.9.

Example 1h

[4-(t-Butoxy]-2R-isobutylsuccinyl]-L-4-oxymethylcarboxybenzyl)-phenylalanine-N-methylamide

[4-(t-Butoxy)-2R-isobutylsuccinyl)-L-(4-hydroxy)phenylalanine-N-methylamide (2.69 g, 6.6 mmol) was taken up in dryacetone (150 ml). Anhydrous Na₂ CO₃ (0.84 g, 7.9 mmol) was added withstirring, followed by dropwise addition of benzyl-2-bromoacetate (2.27g, 9.9 mmol). The reaction flask was flushed with argon and then thereaction mixture heated under reflux. After 48 hours the solvent wasremoved under vacuum. The residue was taken up in CH₂ Cl₂ (100 ml)washed with saturated Na₂ CO₃ (100 ml), 1M HCl (100 ml) and brine (100ml), dried over MgSO₄ and the CH₂ Cl₂ removed under vacuum to give ayellow oil. Flash chromatography (flash silica, 2% MeOH/CH₂ Cl₂) gavethe title compound as a white solid (1.91 g, 52%).

C₃₁ H₄₂ N₂ O₇ MWt=554.69 delta_(H) (250 MHz, CDCl³) 7.36 (5H, s,CH-23-27), 7.14 (2H, d, J=8.7 Hz, CH-9, 11 ), 6.83 (2H, d, J=8.7 Hz,CH-8, 12), 6.33 (1H, d, J=7.9 Hz, CONH), 5.92 (1H, m, CONHMe), 5.24 (2H,s, CH₂ -21), 4.64 (2H, s, CH₂ -19), 4.48 (1H, m, CH-5), 3.08 (1H, dd,J=6.2 Hz, CH₂ -6a), 2.96 (1H, dd, J=7.9 Hz, CH₂ -6b ), 2.68 (3H, d,J=4.8 Hz, CH₃ -13), 2.63 (1H, m, CH-3, 2.46 (2H, m, CH₂ -2a,2b), 1.48(2H, m, CH₂ -15), 1.43 (9H, s, CH₃ -29, 30, 31), 0.87, 0.84 (6H, 2d,J=6.5 Hz, CH₃ -17,18).

Example 1i

[4-Hydroxy-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxybenzyl)-phenylalanine-N-methylamide

[4-(t-Butoxy)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxybenzyl)-phenylalanine-N-methylamide(2.12 g, 3.8 mmol) was taken up in 95% TFA/H₂ O(50 ml ). The solutionwas stirred at 0° C. for 3 hours. TFA/H₂ O removed under vacuum. Theresidue was taken up in CH₂ Cl₂ (50 ml) washed with brine(3×50 ml) driedover MgSO₄ and the solvent removed under vacuum to give the titlecompound as a white solid (1.89 g, 99%).

Example 1j

[4-(N-benzyloxyamino)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxybenzyl)-phenylalanine-N-methylamide

[4-Hydroxy-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxy--benzyl)-phenylalanine-N-methylamide(1.89 g, 3.79 mmol), was dissolved in CH₂ Cl₂ (20 ml). To the solutionwas added HOBT (0.63 g, 4.17 mmol), WSCDI (1.09 g, 5.6 mmol), NMM (0.58g, 5.6 mmol) and after 15 minutes benzylhydroxylamine (0.51 g, 4.17mmol). The reaction mixture was stirred at room temperature. After 16hours the solvent was removed. The yellow residue was taken up in ethylacetate whereupon white crystals precipitated out, which were collectedby filtration and wasted with ethyl acetate to yield the title compoundas a white solid (0.58 g, 27%).

C₃₄ H₄₁ N₃ O₇ MWt=603.72 delta_(H) (250 MHz, CDCl₃) 8.59, (1H, m,CONHOBz), 7.37 (10H, s, CH-23-27, CH-30 to 34), 7.11 (2H, d, J=8.6 Hz,CH-9, 11), 6.80 (2H, d, J=8.6 Hz, CH-8,12), 6.47 (1H, m, CONH), 5.95(1H, m, CONHMe), 5.21 (2H, s, CH₂ -21), 4.87 (2H, m, CH₂ -28), 4.63 (2H,s, CH₂ -19), 4.52 (1H, m, CH-5), 3.02 (2H, m, CH₂ -6a, 6b), 2.71 (3H,d+m, J=4.8 Hz, CH₃ -13, CH-3), 2.42 (2H, m, ,CH₂ -2a, 2b), 1.46 (2 H, m,CH2-15), 1.18 (1H, m, CH-16), 0.87, 0.84 (6H, 2d, J=6.8, 6.6 Hz, CH₃-17,18).

Example 1k

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4oxymethylcarboxylicacid)-phenylalanine-N-methylamide

[4-(N-benzyloxyamino)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxybenzyl)-phenylalanine-N-methylamide(467 mg, 0.77 mmol) was taken up in 10% cyclohexene/ethanol (40 ml) and20% Pd/charcoal (93 mg) added with stirring. The solution was heatedunder reflux and after 3 1/2 hours the solution filtered through glassfibre paper. The filtrate was concentrated down under reduced pressureto give the title compound as a white solid (322 mg, 98%).

mpt=168° C.

Analysis calculated for C₂₀ H₂₉ N₃ O₇ MWt=423.7 Requires C 56.73 H 6.90N 9.92 Found C 56.52 H 6.91 N 9.59 delta_(H) (250 MHz, MeOD) 7.89 (1H,bd, CONHMe), 7.11 (2H, d, J=8.4 Hz, CH-9,11), 6.81 (2H, d, J=8.4 Hz,CH-8.12), 4.56 (2H, s, CH₂ -19), 4.44 (1H, m, CH-5), 3.05 (1H, dd,J=6.4, 6.3 Hz, CH₂ -6a), 2.87 (1H, dd, J=8.8, 9.0 Hz, CH₂ -6b), 2.64(3H, s, CH₃ -13), 2.78-2.58 (1H, bm, CH-3), 2.16 (1H, dd, J=4.7, 7.8 Hz,CH-2a), 2.04 (1H, dd, J=6.5, 6.6 Hz, CH-2b), 1.36 (2H, m, CH₂ -15), 1.13(1H, m, CH-16), 0.81, 0.77 (6H, 2d, J=6.3 Hz, CH₃ -17, 18). delta_(C)(250 MHz, DMSO) 173.34, 170.93, 169.80, 167.17, 155.8, 130.2, 129.5,113.5, 64.1, 53.7, 40.4, 40.2, 40.0, 38.03, 35.86, 35.16, 25.10, 24.63,22.78, 21.41.

Example 2

4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4-oxymethycarboxy-N-methylamide)phenylalanine-N-methylamide

Example 2a

[4-(t-Butoxy)-2R-isobutylsuccinyl]-L-(4-oxymethylcaboxylicacid)-phenylalanine-N-methylamide

Utilising the procedure described in example 1 g but employing[4-(t-Butoxy)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxybenzyl)-phenylalanine-N-methylamide(from example 1h, 9.90 g, 17.85 mmol) in lieu of[4-(t-Butoxy)-2R-isobutylsuccinyl]-L-(4-benzyloxy)-phenylalanine-N-methylamideyielded, the title compound as a white solid (8.07 g, 97.3%)

delta_(H) (250 MHz, CDCl₃) 7.07 (2H, d, J=8.6 Hz, CH-9,11), 7.03 (1H, m,CONHCH), 6.79 (2H, d, J=8.6 Hz, CH-8,12), 6.70 (1H, m, CONHMe), 4.63(1H, m, CH-5), 4.60 (2H, s, CH₂ -19), 2.96 (2H,d, J=7.1 Hz, CH₂ -6),2.66 (3H, d, J=4.8 Hz, CH₃ -13), 2.65 (1H, s, CH-3), 2.65 (1H, s, CH-3),2.43 (1H, dd, J=8.5 Hz, CH₂ -2a), 2.33 (1H, dd, J=5.3 Hz, CH₂ -2b), 1.45(2H, m, CH₂ -15), 1.41 (9H, s, CH₃ -21, 22, 23), 1.20 (1H, m, CH- 16),0.82 (6H, dd, J=6.3, 6.2 Hz, CH₃ -17,18). delta_(C) (250 MHz, CDCl₃)174.1, 170.7, 170.2, 155.3, 128.9, 128.4, 113.3, 79.6, 76.1-75.1, 63.7,53.4, 39.8, 39.6, 36.7, 35.8, 26.6, 24.8, 24.2, 21.3, 20.8.

Example 2b

[4(t-Butoxy)-2R-isobutylsuccinyl]-2-(4-oxymethylcarboxy-N-methylamide)-phenylalanine-N-methylamide

[4-(t-Butoxy) -2R-isobutylsuccinyl]-L-(4-oxymethylcarboxylicacid)-phenylalanine-N-methylamide (0.5 g, 1.07 mmol) was dissolved inCH₂ Cl₂ (100 ml). At 0° C. pentafluorophenol (0.4 g, 2.15 mmol), WSCDI(0.26 g, 1.3 mmol) and N-methylmorpholine (0.11 g, 1.1 mmol) were added.After 15 minutes 8M methylamine in ethanol (0.25 g, 2.7 mmol) was addeddropwise. The solution was allowed to warm to ambient temperature andstirred for 12 hours. A white solid of MeNH₂.HCl precipitated out, butwas not collected. The reaction solution was washed with 1M HCl (100ml), 1M Na₂ CO₃ (100 ml) and brine (100 ml). The CH₂ Cl₂ layer was driedover MgSO₄ and the solvent removed under reduced pressure to give thetitle compound as a white solid (0.44 g, 86%).

delta_(H) (250 MHz, CDCl₁₃) 7.19 (2H, d, J=8.6 Hz, CH-9,11), 6.84 (2H,d, J=8.6 Hz, CH-8,12), 6.60 (1H, m, CONHMe), 6.27 (1H, d,J=7.8 Hz,CONH), 5.99 (1H, m, CONHMe), 4.52 to 4.46 (3H, s+q, CH-5, CH₂ -19), 3.09(2H, m, CH₂ -6a,6b), 2.92 (3H, d, J=4.8 Hz, CH₃ -21), 2.72 (3H, d, J=4.8Hz, CH₃ -13), 2.61 (1H, m, CH-3), 2.46 (2H, m, CH₂ -2a, 2b), 1.45 (11H,s+m, CH₃ -23, 24, 25, CH₂ -15) 1.20 (1H, m, CH-16), 0.87, 0.84 (6H, 2d,J=6.3 Hz, CH₃ -17, 18 ).

Example 2c

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxy-N-methylamide)phenylalanine-N-methylamide

The title compound was prepared from[4-(t-Butoxy)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxy-N-methylamide)-phenylalanine-N-methylamideutilising the method described in examples 1i to 1k

mpt=211° C. Analysis calculated for C₂₁ H₃₂ N₄ O₆ MWt=436.51 Requires C57.78 H 7.39 N 12.84 Found C 57.30 H 7.27 N 12.56 delta_(H) (250 MHz,DMSO) 10.39 (1H, s, CONHOH), 8.74 (1H, s, CONHOH), 7.98 (2H, m, CONHMe),7.85 (1H, d, J=4.7 Hz, CONH), 7.12 (2H, d, J=8.5 Hz, CH-9,11), 6.83 (2H,d, J=8.6 Hz, CH-8,12), 4.38 (2H, s, CH-19), 4.32 (1H, m, CH-5), 2.96(1H, dd, J=5.1, 5.0 Hz, CH₂ -6a), 2.74 (1H, dd, J=9.6, 9.7 Hz, CH₂ -6b),2.64 (3H, d, J=4.7 Hz, CH₃ -21), 2.60 (1H, m, CH-3), 2.55 (3H, d, J=4.5Hz, CH₃ -13), 2.05 (1H, dd, J=3.7, 7.0 Hz CH₂ -2a), 1.91 (1H, dd, J=7.5,7.6 Hz, CH₂ -2b), 1.28 (2H, m CH₂ -15), 0.98 (1H, m, CH-16), 0.77, 0.72(6H, 2d, J=6.3 Hz, CH₃ -17,18) delta_(C) (250 MHz, DMSO) 173.3, 170.9,167.6, 167.2, 155.7, 130.5, 129.6, 113.6, 66.7, 53.7, 40.4-36.0, 35.7,35.2, 25.1, 24.9, 24.6, 22.8, 21.4

Example 3

4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxymethyl)-Phenylalanine-N-methylamide

Example 3a

4-(t-Butoxy) -2R-isobutylsuccinyl]-L-(4-oxymethylcarboxymethyl)-phenylalanine-N-methylamide

[4-(t-Butoxy)-2R-isobutylsuccinyl]-L-(4-oxymethyl-carboxylic acid)phenylalanine-N-methylamide (from example 2a, 0.5 g, 1.08 mmol) wasdissolved in CH₂ Cl₂ (20 ml) and cooled to 0° C. A solution ofdiazomethane in ether (3 ml) was added via a pipette until gas evolutionceased and the reaction solution remained a pale yellow colour. After 30minutes the reaction mixture was treated with 10% acetic acid/etheruntil the solution was colourless and washed with brine (30 ml). The CH₂Cl₂ layer was dried and the solvent removed under reduced pressure togive the title compound as a white solid (0.45 g, 87%).

C₂₅ H₃₃ N₂ O₇ MWt=478.69 delta_(H) (250 MHz, CDCl₃) 7.16 (2H, d, J=8.6Hz, CH-9, 11), 6.83 (2H, d, J=8.6 Hz, CH-8,12), 6.35 (1H, d, J=8.0 Hz,CONHI), 5.91 (1H, m, CONHMe), 4.61 (2H, s, CH2-19), 4.49 (1H, q, J=7.9Hz, CH-5), 3.61 (3H, s, CH3-21), 3.06 (1H, dd, J=6.2 Hz, CH2-6a), 2.96,(1H, dd, J=9.9 Hz, CH2-6b), 2.71 (3H, d, J=4.8 Hz, CH₃ -13), 2.59 (1H,m, CH-3), 2.48 (2H, m, CH₂ -2a,2b), 1.53 (2H, m, CH₂ -15), 1.44 (9H, s,CH₃ -23,24,25), 1.25 (1H, m, CH-16), 0.86 (6H, 2d, J=6.4 Hz, CH₃-17,18).

Example 3b

4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxymethyl)phenylalanine-N-methylamide

The title compound was prepared from 4-(t-Butoxy)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxymethyl)phenylalanine-N-methylamide utilising the method described in examples1i and 1k.

mpt=187° C. Analysis calculated for C₂₁ H₃₁ N₃ O₇ MWt=437.5 Requires C57.65 H 7.14 N 9.60 Found C 57.63 N 7.11 N 9.27 delta_(H) (250 MHz,DMSO) 10.40 (1H, s, CONHOH), 8.76 (1H, s, CONHOH), 7.99 (1H, d, J=8.0Hz, CONH), 7.86 (1H, m, CONHMe), 7.12 (2H, d, J=8.4 Hz, CH-9,11), 6.81(2H, d, J=8.4 Hz, CH-8,12), 4.73 (2H, s, CH₂ -19), 4.33 (1H, m, CH-5),3.69 (3H, s, CH₃ -21), 2.97 (1H, dd, J=4.6 Hz, CH₂ -6a), 2.76 (1H, dd,J=9.9, 10.1 Hz, CH₂ -6b) 2.57, 3H, d, J= 4.2 Hz, CH₃ -13), 2.62-2.56(1H, m, CH-3), 2.07 (1H, dd, J=6.9, 6.8 Hz, CH₂ -2a), 1.93 (1H, dd,J=7.4, 7.6 Hz, CH₂ -2b), 1.29 (2H, m, CH₂ -15), 1.03 (1H, m, CH-16),0.78, 0.74 (6H, 2d, J=6.2 Hz, CH₃ -17,18). delta_(C) (250 MHz, DMSO)173.3, 170.9, 166.6, 167.2, 155.6, 130.5, 129.6, 113.6, 64.2, 53.7,51.3, 40.3-38.1, 35.8, 35.1,25.1, 24.6, 22.7, 21.4

Example 4

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxy-N-benzylamide)-phenylalanine-N-methyl-amide

Example 4a

[4-(t-Butoxy)-2R-isobutylsuccinyl]-L-(4oxymethylcarboxy-N-benzylamide)-phenylalanine-N-methylamide.

Utilising the procedure described in example 2b employing benzylamine(0.25 g, 2.4 mmol) in lieu of methylamine yielded the title compound asa white solid (0.53 g, 81%).

C₂₉ H₄₁ N₃ O₆ MWt=527 delta_(H) (250 MHz, CDCl₃ 7.40-7.27 (5H, m, CH-23to 27), 7.17 (2H, d, J=8.6 Hz, CH-9,11), 6.92 (1H, m, CONHBz), 6.85 (2H,d, J=8.6 Hz, CH-8,12), 6.29 (1H, d, J=7.8 Hz, CONH), 5.96 (1H, m,CONHMe), 4.56-4.45 (5H, m, CH-5, CH₂ -21, CH₂ -19), 3.04 (2H, m, CH₂-6), 2.70 (3H, d, J=4.8 Hz, CH₃ -13), 2.58 (1H, m, CH-3), 2.43 (1H, dd,J=8.5 Hz, CH₂ -2a), 2.33 (1H, dd, J=5.3 Hz, CH₂ b), 1.45 (2H, m, CH₂-15), 1.41 (9H, s, CH₃ -23,24,25), 1.20 (1H, m, CH-16), 0.86 (6H, 2d,J=6.3, 6.2 Hz, CH₃ -17,18).

Example 4b

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxy-N-benzylamide)-phenylalanine-N-methylamide

The title compound was prepared from [4-(t-Butoxy)-2R-isobutylsuccinyl]-L-(4-oxymethylenecarboxy-N-benzylamide)phenylalanine-N-methylamide utilising the method described in examples1i to 1k.

mpt=206° C. Analysis calculated for C₂₇ H₃₆ N₄ O₆ MWt =512.6 Requires C63.26 H 7.08 N 10.93 Found C 62.52 H 7.09 N 10.97 delta_(H) (250 MHz,DMSO) 10.41 (1H, s, CONHOH), 8.76 (1H, s, CONHOH), 8.62 (1H, t, J=6.1Hz, CONHCHPh), 8.01 (1H, d, J=8.3 Hz, CONH), 7.87 (1H, m, CONHMe), 7.26(5H, m, CH-23 to 27), 7.13 (2H, d, J=8.5 Hz, CH-9,11), 6.86 (2H, d,J=8.5 Hz, CH-8,12), 4.48 (2H, S, CH₂ -19), 4.34 (2H, d, J=6.0 Hz, CH₂-21), 2.97 (1H, dd, J=4.5, 4.8 Hz, CH₂ -6a), 2.76 (1H, dd, J=9.5, 9.6Hz, CH₂ -6b), 2.64-2.55 (1H, bm, CH-3), 2.56 (3H, d, J=4.4 Hz, CH₃ -13)2.6 (1H, dd, J=3.2, 7.1 Hz, CH₂ -2a), 1.92 (1H, dd, J=7.6 Hz, CH₂ -2b),1.28 (2H, m, CH₂ -15), 0.97 (1H, m, CH-16), 0.78, 0.73 (6H, 2d, J=6.3Hz, CH₃ -17,18). delta_(C) (250 MHz, DMSO) 173.5, 170.9, 167.3, 167.2,155.7, 138.6, 130.5, 129.6, 127.8, 126.8, 126.3, 113.9, 66.6, 53.7,41.32, 40.1-38.1, 35.2, 25.1, 24.7, 22.8, 21.4

Example 5

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxy-beta-alanine)phenylalanine-N-methylamide

Example 5a

[4-(t-Butoxy)-2R-isobutylsuccinyl]-L-(4-oxymethyl-carboxy-beta-alaninebenzyl ester) phenylalanine-N-methylamide

Utilising the procedure described in example 2b employing beta-alaninebenzyl ester (0.76 g, 2.16 mmol) in lieu of methylamine yielded thetitle compound as a yellow oil (0.65 g, 97%).

C₃₄ H₄₇ N₃ O₈ MWt=625 delta_(H) (250 MHz, CDCl₃) 7.35 (5H, s, CH-26 to30), 7.17 (2H, d, J=8.5 Hz, CH-9,11), 7.19 (1H, m, CONHCH₂), 6.82 (2H,d,J=8.5 Hz, CH-8,12), 6.33 (1H, d, J=7.8 Hz, CONH), 6.01 (1H, m, CONHMe),5.13 (2H, s, CH₂ -24), 4.49 (1H, q, J=6.7, 7.6 Hz, CH-5), 4.44 (2H, s,CH₂ -19), 3.64 (2H, q, J=6.1 Hz, CH₂ -21), 3.06 (2H, m, CH₂ -6a, 6b),2.72 (3H, d, J=4.8 Hz, CH₃ -13), 2.66-2.62 (3H, m, CH2-22, CH-3),2.50-2.39 (2H, m, CH₂₂ a,b), 1.48 (2H, m, CH₂ -15), 1.22, (1H, m,CH-16). 0.87, 0.84 (6H, 2d, J=6.4, 6.3 Hz, CH₃ -17, 18).

Example 5b

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxy-beta-alanine)phenylalanine-N-methylamide

The title compound was prepared from[4-(t-butoxy)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxy-beta-alaninebenzyl ester)phenyl alanine-N-methylamide utilising the method describedin examples 1i to 1k.

mpt=195° C. Analysis calculated for C₂₃ H₃₄ N₄ O₈ MWt -494.6 Requires C55.86 H 6.93 N 11.33 Found C 55.94 H 6.96 N 11.51 delta_(H) (250 MHz,MeOD) 7.13 (2H, d, J=8.5 Hz, CH-9,11), 6.86 (2H, d, J=8.5 Hz, CH-8,12),4.47-4.42 (1H, m, CH-5), 4.42 (2H, s, CH₂ -19), 3.48 (2H, t, J=6.7 Hz,CH₂ -21), 3.06 (1H, dd, J=6.4, 6.3 Hz, CH₂ -6a), 2.85 (1H, dd, J=8.7,8.8 Hz, CH₂ -6b), 2.80-2.64 (1H, m, CH-3), 2.64 (3H, s, CH₃ -13), 2.50(2H, t, J=6.7 Hz, CH₂ -22), 2.17 (1H, dd, J=6.5 Hz, CH₂ -2a), 2.04 (1H,dd, J=6.5 Hz, CH₂ -2b) 1.36 (2H, m, CH₂ -15), 1.06 (1H, m, CH-16), 0.81,0.77 (6H, 2d, J=6.3 Hz, CH₃ -17,18). delta_(C) (250 MHz, MeOD) 177.0,175.5, 173.9, 171.1, 170.6, 157.9, 132.0, 131.4, 115.8, 68.3, 56.2,50.0, 49.6-47.9, 42.5, 42.4, 37.9, 36.8, 36.0, 34.6, 26.8, 26.3, 23.5,22.

Example 6

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4-oxymethyl carboxy-glycinemethyl ester) phenylalanine-N-methylamide

Example 6a

[4-(t-Butoxy)-2R-isobutylsuccinyl]-L-4-oxymethyl carboxy-glycinemethylester)-phenylalanine-N-methylamide.

Utilising the procedure described in example 2b employing glycine methylester hydrochloride (0.13 g, 1.07 mmol) in lieu of methylamine yieldedthe title compound as a white solid (0.45 g, 78.5%).

C₂₇ H₄₁ N₈ O₈ MWt=535.64 delta_(H) (250 MHz, CDCl₃) 7.17 (2H, d, J=8.6Hz, CH-9,11), 7.15 (1H, m, CH₂ CONHCH₂), 6.83 (2H, d, J=8.6 Hz, CH-8),6.45 (1H, d, J=8.0 Hz, CHCONHCH), 6.26 (1H, m, CONHMe), 4.53 (1H, m,CH-5), 4.48 (2H, s, CH₂ -19), 4.13 (2H, d, J=6.3 Hz, CH₂ -21), 3.76 (3H,s, CH₃ -23), 3.04 (2H, m, CH₂ -6), 2.72 (3H, d, J=4.8 Hz, CH₃ -13), 2.51(1H, m, CH-3), 2.42 (2H, m. CH₂ -2), 1.47 (2H, m, CH₂ -15), 1.43 (9H, s,CH₃ -24,25,26), 1.19 (1H, m, CH-16), 0.83 (6H, dd, J=6.3 Hz, CH3-17,18).

Example 6b

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4-oxymethyl carboxy-glycinemethyl ester)-phenylalanine-N-methylamide

The title compound was prepared from4-(t-Butoxy)-2R-isobutylsuccinyl]-L-4-oxymethyl carboxy-glycine methylester)-phenylalanine-N-methylamide utilising the method described inexamples 1i to 1k.

mpt =180°-185° C. Analysis calculated for C₂₃ H₃₄ N₄ O₈ MWt=494.55Requires C 55.86 H 6.93 N 11.33 Found C 53.45 H 6.83 N 11.50 delta_(H)(250 MHz, MeOD) 7.14 (2H, d, J 8.6 Hz, CH-9,11) 6.89, (2H, d, J=8.6 Hz,CH-8,12), 4.49 (2H,s, CH₂ -19), 4.44 (1H, m, CH-5), 3.99 (2H, s, CH₂-21), 3.69 (3H, s, CH₃ -23), 3.11-2.81 (2H, m, CH₂ 6), 2.72-2.63 (1H, m,CH-3), 2.64 (3H, s, CH₃ -13), 2.21 (1H,dd, J=7.8 Hz, CH₂ -2a), 2.04 (1H,dd, J=6.7,6.6 Hz, CH₂ -2b), 1.35 (2H, m, CH₂ -15) 1.05 (1H, m, CH-16),0.79 (6H, dd, J=6.4 Hz, CH₃ -17,18). delta_(C) (250 MHz, MeOD) 177.1,173.9, 171.8, 171.6, 170.6, 158.0, 132.1, 131.4, 115.9, 68.3, 56.3,52.7, 50.0-48.0, 42.6, 42.4, 41.5, 37.9, 36.8, 26.8, 26.3, 22.3

Example 7

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-4-oxymethylcarboxymethyl)-phenylalanine-N-methylamide.

Example 7a

[4-(t-Butoxy)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxymethyl-phenylalanine-N-methylamide.

When the procedure described in example 1 g was utilised employing[4-(t-butoxy)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxybenzyl ester)phenylalanine-N-methylamide (from example 1 h 9.10 g, 16.41 mmol) inlieu of [4-(t-Butoxy)-2R-isobutylsuccinyl]-L-(4-benzyloxy)phenylalanine-N-methylamide a transesterification reaction occurred andan ethyl group was transferred from the solvent to the reactantmolecule. The reaction yielded after chromatography (flash silica, 100%ethyl acetate) the title compound as a white solid (0.51 g, 6.3%)

C₂₆ H₄₆ N₂ O₇ MWt=492.62 ¹ Hnmr delta_(H) (250 MHz, CDCl₃) 7.09 (2H, d,J=8.6 Hz, CH-9,11), 6.77 (2H, d, J=8.6 Hz, CH-8,12), 6.68 (1H, d, J=8.8Hz, CONHCH), 6.57 (1H, m, CONHMe), 4.55 (1H, m, CH-5), 4.53 (2H, s, CH₂-19), 4.21 (2H, q, J=7.1 Hz, CH₂ -21), 2.97 (2H, m, CH₂ -6), 2.72-2.52(1H, m, CH-3), 2.65 (3H, d, J=4.8 Hz, CH₃ -13), 2.43 (1H, dd, J=7.8 Hz,CH₂ -2a), 2.27 (1H, dd, J=6.8 Hz, CH₂ -2b), 1.42 (2H, m, CH₂ -15), 1.37(9H, s, CH₃ -23,24,25), 1.25 (3H, t, J=7.1 Hz, CH₃ -22), 1.14 (1 H, m,CH-16), 0.78 (6H, dd, J=6.4 Hz, CH₃ -17,18).

Example 7b

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxymethyl)-phenylalanine-N-methylamide.

The title compound was prepared from[4-(t-butoxy)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxyethyl)-phenylalanine-N-methylamideutilising the method described in examples 1i to 1k.

mpt 185° C. Analysis calculated for C₂₂ H₃₃ N₃ O₇ MWt=451.52 Requires C58.52 H 7.37 N 9.31 Found C 58.54 H 7.28 N 9.26 delta_(H) (250 MHz,MeOD) 7.11 (2H, d, J=8.6 Hz, CH-9,11), 6.79 (2H,d, J=8.6 Hz, CH-8,12),4.61 (2H, s, CH₂ -19), 4.43 (1H, m, CH-5), 4.19 (2H, q, J=7.1, 7.2Hz,CH₂ -21), 3.05 (1H, dd, J=6.4, 6.4 Hz, CH₂ -6a), 2.81 (1H, dd, J=9.1,8.9 Hz, CH₂ -6b), 2.64 (3H, s, CH₃ -13), 2.78-2.58 (1H, m, CH-3), 2.12(1H, dd, J=7.8, 7.9 Hz, CH₂ -2a), 2.06 (1H, dd, J=6.7, 6.8 Hz, CH₂ -2b),1.31 (2H, m, CH₂ -15), 1.24 (3H, t, J=7.1 Hz, CH₃ -22) 1.09 (1H, m,CH-16), 0.79 (6H, dd, J=6.4 Hz, CH₃ -17, 18). delta_(C) (250 MHz, MeOD)177.2, 174.0, 171.0, 170.6, 158.1, 131.8, 131.2, 115.6, 66.4, 62.4,56.4, 50.1,-48.0, 42.6, 42.4, 38.1, 36.9, 26.8, 26.4, 23.6, 22.4, 14.

Example 8

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxyglycine)Phenylalanine-N-methylamide

Example 8a

[4-(t-Butoxycarbonyl)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxyglycinebenzyl ester)-phenylalanine-N-methylamide

Utilising the procedure described in example 2b employing glycine benzylester hydrochloride (0.24 g, 1.21 mmol) in lieu of methylamine yieldedthe title compound as a white solid (0.55 g, 89.5%).

C₃₃ H₄₅ N₃ O₈ MWt=611.74 delta_(H) (250 MHz, CDCl₃) 7.38 (5H, m, CH-24to 29), 7.22-7.11 (1H, m, CONHCH₂ CO₂ Bz), 7.17 (2H, d, J=7.76 Hz, CH-9,11), 6.84 (2H, d, J=7.6 Hz, CH-8, 12), 6.47 (1H, d, J=8.1 Hz, CHCONHCH),6.24 (1H, m, CONHMe), 5.19 (2H, s, CH₂ -23), 4.62-4.42 (1H, m,CH-5),4.48 (2H, s, CH₂ -19), 4.16 (2H, d, J=6.5 Hz, CH₂ -21), 3.05 (2H, m, CH₂-6), 2.77-2.55 (1H, m, CH-3), 2.73 (3H, d, J=4.2 Hz, CH₃ -13), 2.53 (1H,dd, J=6.6 Hz, CH₂ -2a), 2.35 (1H, dd, J=5.3 Hz, CH₂ -2b), 1.55-1.37 (2H,m, CH₂ -15), 1.45 (9H, s, CH₃ -30,31,32), 1.22, (1H, m, CH-16), 0.97(6H, m, CH₃ -17, 18).

Example 8b

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxyglycine)phenylalanine-N-methylamide

The title compound was prepared from [4-(t-butoxycarbonyl)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxyglycine benzylester)-phenylalanine-N-methylamide utilising the method described inexamples 1i to 1k.

mpt=142.5° C. Analysis calculated for C₂₂ H₃₂ N₄ O₈ MWt=480.52 RequiresC 54.99; H 6.71 N 11.66 Found C 54.19; H 6.87 N 10.96 delta_(H) (250MHz, DMSO) 10.42 (1H, s, CONHOH), 8.32 (1H, m, CONHCH₂ CO₂ H), 8.01 (1H,d, J=8.1 Hz, CHCONHCH), 7.87 (1H, m, CONHMe), 7.13 (2H, d, J=7.6Hz,CH-9,11), 6.87 (2H, d, J=7.3 Hz, CH-8,12), 4.46 (2H, s, CH₂ -21), 4.33(1H, m, CH-5), 3.80 (2H, d, J=5.5 Hz, CH₂ -19), 2.96 (1H, dd, J=4.6, 4.5Hz, CH₂ -6a), 2.75 (1H, m, CH₂ -6b), 2.56 (3H, s, CH₃ -13), 2.68-2.43(1H, m, CH-3), 2.05 (1H, dd, J=7.0, 6.6 Hz, CH₂ -2a), 1.91 (1H, dd,J=5.3 Hz, CH₂ -2b), 1.26 (2H, m, CH₂ -15), 1.05 (1H, m, CH-16), 0.75(6H, dd, J=5.7 Hz, CH₃ -17,18). delta_(C) (250 MHz, DMSO) 173.4, 170.9,170.5, 167.6, 167.2, 155.6, 130.6, 129.6, 113.9, 66.5, 53.7, 40.3, 40.2,39.7-38.4, 5.9, 35.2, 25.9, 25.1, 22.8, 21.4

Example 9

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxy-N,N-dimethylamide)-phenylalanine-N-methylamide

Example 9q

[4-(t-Butoxy)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxy-N,N-dimethylamide)-phenylalanine-N-methylamide.

Utilising the procedure described in example 2b employing dimethylaminehydrochloride (0.11 g, 1.30 mmol) in lieu of methylamine yielded thetitle compound as (a white solid (0.49 g, 92.3%).

C₂₆ H₄₁ N₃ O₆ MWt=491.63 delta_(H) (250 MHz, CDCl₃) 7.13 (2H, d, J=8.5Hz, CH-9,11), 6.85 (2H, d, J=8.5 Hz, CH-8,12), 6.42 (1H, d, J=8.0 Hz,CHCONHCH), 6.19 (1H, m, CONHMe), 4.63 (2H, s, CH₂ -19), 4.50 (1H, m,CH-5), 3.12-2.82 (2H, m, J=2-6), 3.07 (3H, s, CH₃ -21), 2.96 (3H, s, CH₃-22), 2.68 (3H, d, J=4.5 Hz, CH₃ -13), 2.62 (1H, m, CH-3), 2.52 (1H, dd,J=8.5 Hz, CH₂ -2a), 2.33 (1H, dd, J=4.5 Hz, CH₂ -2b), 1.55-1.35 (2H, m,CH₂ -15), 1.42 (9H, s, CH₃ -23,24,25), 1.19 (1H, m, CH-16), 0.83 (6H,dd, J=6.3 Hz, CH₃ -17,18).

Example 9b

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxy-N,N-dimethylamide)-phenylalanine-N-methylamide

The title compound was prepared from[4-(t-Butoxy)-2R-isobutylsuccinyl]-L-(4-oxymethyl-carboxy-N,N-dimethylamide)-phenylalanine-N-methylamideutilising the method described in examples 1i to 1k.

mpt 197° C. Analysis calculated for C₂₂ H₃₄ N₄ O₆ MWt=450.54 Requires C58.65 H 7.61 N 12.44 Found C 58.58 H 7.54 N 12.33 delta_(H) (150 MHz,DMSO) 0.42 (1H, s, CONHOH), 8.77 (1H, s, CONHOH), 7.99 (1H, d, J=8.0 Hz,CHCONHCH), 7.87 (1H, m, CONHMe), 7.09 (2H, d, J=8.5 Hz, CH-9,11), 6.78(2H, d, J=8.5 Hz, CH-8,12), 4.71 (2H, S, CH₂ -19), 4.32 (1H, m, CH-5),2.98 (3H, s, CH₃ -21), 2.94 (1H, m, CH₂ -6a), 2.83 (3H, s, CH₃ -22),2.73 (1H, m, CH₂ -6b), 2.55 (3H, m, CH₃ -13), 2.68-2.45 (1H, m, CH-3),2.01 (1H, dd, J=6.8 Hz, CH₂ -2a), 1.91 (1H, dd, J=6.8 Hz, CH₂ -2b), 1.28(2H, m, CH₂ -15), 0.99 (1H, m, CH-16), 0.74 (6H, dd, J=6.3, 6.2 Hz, CH₃-17,18). delta_(C) (250 MHz, CDCl₃) 173.4, 170.9, 167.2, 166.8, 156.1,130.0, 129.4, 113.7, 65.5, 53.7, 40.9-38.0, 35.9, 35.2, 34.5, 25.1,24.6, 21.4

Example 10

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl](4-acetamido)phenylalanine-N-methylamide

Example 10a

p-Aminophenylalanine methyl ester

To a stirred solution of p-aminophenylalanine (2.05 g, 9.9 mmol) inmethanol (100 ml) at 0° C. was added thionyl chloride (20 ml) dropwise.Upon complete addition the reaction mixture was heated under reflux.After 24 hours the reaction mixture was cooled and reduced underpressure to give a brown oil. The brown oil was taken up in ethylacetate (200 ml) and washed with saturated sodium bicarbonate solution(200 ml). The aqueous layer was further extracted with ethyl acetate(5×200 ml). The combined organic layer was dried over MgSO₄ and reducedunder pressure to yield the title compound as a brown solid (1.7 g,88.5%).

delta_(H) (250 MHz, CDCl₃) 6.95 (2H, d, Ar-H), 6.60 (2H, d, Ar-H, 3.70(3H, s, CO₂ CH₃), 3.65 (1H, dd, CHCO₂ CH₃), 2.95 (1H, dd, CH₂ Ph), 2.75(1H, dd, CH₂ Ph), 2.60 (4H, bm, 2NH2).

Example 10b

P-Aminophenylalanine-N-methylamide

To a stirred solution of P-aminophenylalanine methyl ester (1.7 g, 8.8mmol) in ethanol (150 ml) was added methylamine in ethanol (33%/8M) (100ml). After stirring at room temperature for 24 hours the reactionmixture was reduced under pressure to yield the title compound as abrown solid (1.7 g, 100%) delta_(H) (250 MHz, CDCl₃) 6.90 (2H, d, Ar-H),6.55 (2H, d, Ar-H), 3.40 (1H, dd, CHCONHCH₃), 3.00 (1H, dd, CH₂ Ph) 2.70(3H, d, CONHCH₃), 250 (1H, dd, CH₂ Ph).

Example 10c

[4-(t-Butoxy)-2R-isobutylsuccinyl]-L-4-amino-phenylalanine-N-methylamide

Utilising the procedure described in example 1f but employingp-aminophenylalanine-N-methylamide (1,7 g, 8.79 mmol) in lieu ofO-benzyl tyrosine-N-methylamide yielded the title compound as a lightbrown oil (1.39 g, 38%)

delta_(H) (250 MHz, CDCl₃) 7.00 (2H, d, J=8.3 Hz, CH-9,11), 6.60 (2H, d,J=8.3 Hz, CH-8, 12), 6.45 (1H, d, J=7.8 Hz, CONHCH), 6.10 (1H, d, J=4.7Hz, CONHMe), 4.50 (1H, q, J=7.9Hz, CH-5), 2.95 (2H, dq, J=13.7, 7.91 Hz,CH₂ -2), 2.67 (3H, d, J=4.9 Hz, CH₃ -13, 260 (1H, m, CH-3), 2.45 (2H,dq, J=16.3, 5.1 Hz, CH₂ -6), 1.45 (2H, m, CH₂ -15), 1.40 (9H, s,C(CH₃)₃), 1.20 (1H, m, CH-16), 0.85 (6H, dd, J=6.3, 7.9 Hz, CH₃ -17,18). deltaC (250 MHz, CDCl₃) 174.6, 171.6, 171.5, 145.1, 130.1, 126.6,115.3, 80.9, 54.9, 41.0, 38.2, 37.4, 28.0, 26.0, 25.6, 22.6, 22.3

Example 10d

[4-(t-Butoxy)-2R-isobutylsuccinyl]-L-4-acetamido)phenylalanine-N-methylamide

A solution of [4-(t-Butoxy)-2R-isobutylsuccinyl-L-(4-aminophenylalanine-N-methylamide (0.7 g, 1.73 mmol), acetic anhydride (0.2 g,0.18 ml , 1.9 mmol), DMAP (catalytic amount) and triethylamine (0.2 g,0.3 ml, 1.9 mmol) in CH₂ Cl₂ (10 ml) was stirred at room temperature.After 24 hours the reaction mixture was washed with 2M HCl (20 ml×2) andbrine (20 ml). The CH₂ Cl₂ layer was dried over MgSO₄ and reduced underpressure to yield the title compound as a light brown solid (0.77 g,99.4%).

delta_(H) (250 MHz, CDCl13) 8.35 (1H, bs, NHCOCH3), 7.40 (2H, d, J=8.4Hz, CH-9,11), 7.10 (2H, d, J=8.4 Hz, CH-8,12), 4.55 (1H, dd, J=7.7, 7.1Hz, CH-5), 2.65 (3H, d, J=4.7 Hz, CH₃ -13), 2.60 (1H, m, CH-3), 2.40(2H, m, CH₂ -2), 1.45 (2H, m, CH₂ -15), 1.40 (9H, s,C(CH₃)₃), 1.15 (1H,m, CH-16), 0;80 (6H, dd, J=6.2, 3.0 Hz, CH₃ -17,18).

Example 10e

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4acetamido)phenylalanine-N-methylamide

The title compound was prepared from[4-(t-Butoxy)-2R-isobutylsuccinyl]-L-(4-acetamido)phenylalanine-N-methylamide utilising the method described in examples1i to 1k.

Analysis calculated C₂₀ H₃₀ N₄ O₅ MWt=406.48 Requires C59.09 H7.44 N13.78 Found C58.90 H7.38 N 13.70 delta_(H) (250 MHz, MeOD) 7.42 (2H, d,J=8.4 Hz, CH-9,11), 7.13 (2H, d, J=8.4 Hz, CH-8,12), 4.46 (1H, dd,J=9.0, 6.4 Hz, CH-5), 3.10 (1H, dd, J=13.8, 9.1 Hz, CH₂ -6a), 2.86 (1H,dd, J=13.8, 9.1 Hz, CH₂ -6b), 2.65 (3H, s, CH₃ -13), 2.60 (1H, m, CH-3),2.19 (1H, dd, J=14.5, 6.5 Hz, CH₂ -2a), 2.05 (3H, s, NHCOCH₃), 2.04 (1H,dd J=14.5, 6.5 Hz, CH₂ -2b), 1.40 (1H, m, CH₂ -15a), 1.30 (1H, m,CH-16), 1.05 (1H, m, CH₂ -15b), 0.77 (6H, dd, J=6.4, 5.4 Hz, CH₃-17,18). delta_(C) (250 MHz, MeOD) 177.1, 173.9, 171.5, 170.7, 138.5,134.5, 130.6, 121.1, 42.7, 42.4, 38.1, 36.8, 26.7, 26.3, 23.7, 23.5,22.3

Example 11

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4-cyano)phenylalanine-N-methyl amide

Example 11a

N-(N-dimethylthioimidate)-L-(4-cyano)phenylalaninyl)-2R-bornane-10,2-sultam

To a solution ofN-((N-dimethylthioimidate)glycinyl)-(2R)-bornane-10,2-sultam (Oppolzer'schiral glycine equivalent, 10 g, 26.6 mmol) in dry THF (50 ml) underargon atmosphere at -65° C. was added n-butyllithium (1.36M, 19.5 ml,26.6 mmol). After stirring the reaction mixture at -65° C. for 30 mins(20 ml) alpha-bromotolunitrile (5.21 g, 26.6 mmol) in dry THF (20 ml)was added via syringe followed by addition of HMPA (20 ml) via cannula.The reaction mixture was kept at -65° C. for 1 hour, then allowed towarm to ambient temperature overnight. The reaction mixture was quenchedwith saturated NH₄ Cl solution (5 ml) and the THF removed underpressure. The residue was dissolved in ethyl acetate and washed with0.5M HCl (50 ml) and H₂ O(2×50 ml). The ethyl acetate layer was driedover MgSO₄ and the solvent removed under pressure to yield the titlecompound as a yellow solid (0.7 g, 93%).

C₂₃ H₂₉ N₃ O₃ S₃ MWt=491.67 delta_(H) (250 MHz, CDCl₃) 7.56 (2H, d,J=8.2 Hz, Ar-H), 7.41 (2H, d, J=8.2 Hz, Ar-H), 5.18 (1H, dd, J=8.2, 5.6Hz, CHNC(SCH₃)₂) 3.90 (1H, dd, J=8.6, 4.7 Hz, CHNSO₂), 3.46 (2H, dd,J=3.2 Hz, CH₂ SO₂, 3.39 (1H, dd, J=12.7, 5.6 Hz, CH₂ Ph), 3.12 (1H, dd,J=12.7, 8.3 Hz, CH₂ Ph), 2.48 (3H, s, SCH₃) 2.40 (3H, s, SCH₃), 2.13 to1.77 (5H, m, CH₂ +CH₂ +CH), 1.47 to 1.23 (2H, m, CH₂), 0.95 (3H, s,CH₃), 0.93 (3H, s, CH₃). delta_(C) (250 MHz, CDCl₃) 170.14, 163.85,143.00, 131.78, 130.67, 118.93, 110.37, 65.85, 65.23, 53.10, 48.43,47.62, 44.43, 40.23, 38.17, 32.69, 26.30, 20.42, 19.70, 15.25, 14.82.

Example 11b

N-((4-cyano)phenylalaninyl)-(2R)-bornane-10,2-sultam

Hydrochloric acid (0.2M, 125 ml, 250 mmol) was added to a stirredsolution ofN-((N-dimethylthioimidate)-L-(4-cyano)phenylalaninyl)-(2R)-bornane-10,2-sultam(12.8 g, 22.8 mmol) in THF (250 ml). After stirring the reaction mixturefor 24 hours at room temperature, the THF was removed under pressure andthe residue partitioned between water and CH₂ Cl₂. The aqueous layer wasbasified with saturated sodium bicarbonate solution and extracted withethyl acetate (3×100 ml). The combined ethyl acetate layers were driedover MgSO₄ and the solvent removed under pressure to yield the titlecompound as a white solid (3.21 g, 36.3%).

C₂₀ H₂₅ N₃ O₃ S MWt=387.48

Example 11c

N-((N-tertbutoxycarbonyl)-(4-cyano)phenylalaninyl)-(2R)-bornane-10,2-sultam.

A solution of N-((4-cyano)phenylalaninyl)-(2R)-bornane-10,2-sultam (4.60g, 11.9 mmol) in CH₂ Cl₂ (20 ml) was treated with Boc-anhydride (2.58 g,13.0 mmol) and triethylamine (1.32 g, 13.0 mmol). After stirring thereaction mixture for 16 hours at room temperature, it was washed with 1Mcitric acid (2×50 ml). The organic layer was dried over MgSO₄ and thesolvent removed under pressure to yield the title compound as a paleyellow foam (4.46 g, 77%)

C₂₅ H₂₅ N₃ O₅ S MWt=59 delta_(H) (250 MHz, CDCl₃) 7.561 (2H, d, J=8.2Hz, Ar-H), 7.38 (2H, d, J=8.0 Hz, Ar-H, 5.13 (2H, bm, NH+CHNH), 3.88(1H, dd, J=7.1, 5.0 Hz, CHNSO₂), 3.51 (2H, dd, J=2.1 Hz, CH₂ SO₂), 3.38(1H, bdd, CH₂ Ph), 2.80 (1H, bdd, CH₂ Ph) 2.14 to 1.80 (5H, bm, CH₂ +CH₂+CH), 1.43 to 1.17 (11H, bm, C(CH₃)₃ +CH₂), 1.04 (3H, s, CH₃), 01.97(3H, s, CH₃).

Example 11d

N-Boc-4-cyanophenylalanine

A solution of N-((N-tertbutoxycarbonyl)-(4-cyano)phenylalaninyl)-(2R)-bornane-10,2-sultam (8.78 g, 18.0 mmol) in THF (120ml) was treated with a solution of Li(OH)₂ (3.02 gm 72.0 mmol) in H₂ O(60 ml). After stirring -the reaction mixture for 16 hours at roomtemperature the THF was removed under pressure. The residue was taken upin CH₂ Cl₂ (75 ml), the aqueous layer separated and further extractedwith CH₂ Cl₂ (2×75 ml). The aqueous layer was then acidified with 2M HClto pH2 and extracted with ethyl acetate (3×75 ml). The combined ethylacetate layers were dried over MgSO₄ and the solvent removed underpressure to yield the title compound as a pale yellow solid (4.47 g,85.5%).

C₁₅ H₁₈ N₂ O₄ MWt=290.31 delta_(H) (250 MHz, CDCl₃ /DMSO) 7.52 (2H, d,Ar-H) 7.26 (2H, d, Ar-H, 5.16 (1H, bd, NH), 4.48 (1H, dd, CHNH, 3.22(1H, dd, CH₂ Ph), 3.02 (1H, dd, CH₂ Ph), 1.36 (9H, bs, C(C₃)₃).

Example 11e

N-Boc-4-cyanophenylalanine-N-methylamide

A solution of N-Boc-4-cyanophenylalanine (1.92 g, 6.6 mmol) andpentafluorophenol (2.43 g, 13.2 mmol) in CH₂ Cl₂ (30 ml) at 0° C. wastreated with 4-methylmorpholine (0.74 g, 7.3 mmol). After stirring thereaction mixture for 3 hours, 8M methylamine in ethanol (15 ml, 19.8mmol) was added and the reaction mixture left to stir at roomtemperature for 16 hours. The solvent was removed under pressure, theresidue dissolved in CH₂ Cl₂ (50 ml) and washed with saturated sodiumbicarbonate (2×50 ml), 1M citric acid (2×50 ml) and brine (50 ml). TheCH₂ Cl₂ layer was dried over MgSO₄ and the solvent removed underpressure to yield the title compound as a white solid (1.84 g, 92%).

C₁₆ H₂₁ N₃ O₃ MWt=303.35 delta_(H) (250 MHz, CDCl₃) 7.60 (2H, d, J=8.3Hz, Ar-H), 7.33 (2H, d, J=8.4 Hz, Ar-H), 6.00 (1H, bd, NH), 5.04 (1H,bd, CONHCH₃) 4.35 (1H, bd, CHNH), 3.21 (1H, dd, CH₂ Ph), 3.04 (1H, dd,CH₂ Ph), 2.76 (3H, d, J=4.9 Hz CH₃), 1.39 (9H, s, C(CH₃)₃)

Example 11f

4-cyanophenylalanine-N-Methylamide

Utilising the procedure described in example 1e but employingN-Boc-4-cyano-phenylalanine-N-methylamide (3.36 g, 11.1 mmol) in lieu ofN-Boc-O-benzyl tyrosine methylamide yielded the title compound as theTFA salt (3.49 g, 100%).

Example 11g

[4-(t-Butoxy)-2R-isobutylsuccinyl-L-(4-cyano)-phenylalanine-N-methylamide

Utilising the procedure described in example 1f but employing4-cyanophenylalanine-N-methylamide TFA salt (3.4 g, 11.1 mmol) in lieuof O-benzyl tyrosine N-methylamide and 4-methylmorpholine (2.24 g, 22.2mmol) yielded the title compound as an off white solid (3.11 g, 67.5%)

C₂₃ H₃₃ N₃ O₄ MWt=415.51 delta_(H) (250 MHz, CDCl₃) 7.59 (2H, d, J=8.2Hz, CH-9, 11), 7.37 (2H, d, J=8.1 Hz, CH-8, 12), 6.29 (1H, bd, CONH)6.23 (1H, bd, CONHCH₃), 4.60 (1H, dd, CH-5), 3.26 (1H, dd, CH₂ -6a),3.17 (1H, dd, CH₂ -6b), 2.75 (3H, d, J=4.8 Hz, CH₃ -13), 2.49 (1H, dd,CH₂ -2, 2.37 (1H, dd, CH₂ -2b), 1.57 to 1.30 (2H, m, CH₂ -15a+CH-16,1.43 (9H, s, C(CH₃)₃), 1.30 to 1.10 (1H, m, CH₂ -15b) 0.87 (3H, d, J=6.9Hz, CH₃ -17), 0.84 (3H, d, J=7.1 Hz, CH₃ -18).

Example 11h

[4-N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4-cyano)phenylalanine-N-methylamide

The title compound was prepared from4-(t-butoxy)-2R-isobutylsuccinyl]-L-(4-cyano)-phenylalanine-N-methylamideutilising the method described in examples 1i to 1k.

Analysis calculated for C₁₉ H₂₆ N₄ O₄ MWt=374.43 Calculated C 60.94 H7.00 N 14.96 Found 61.18 H 7.21 N 14.14 delta_(H) (250 MH, MeOD) 7.61(2H, d, J=8.2 Hz, CH₂ ⁻ 9, 11), 7.38 (2H, d, J=8.2 Hz, CH₂ -8, 12), 7.05(1H, s, NH), 4.53 (1H, dd, J=9.1, 6.0 Hz, CH-5), 3.23 (1H, dd, J=14.0,5.9 Hz, CH₂ -6b), 3.01 (1H, dd, J=9.1 Hz, CH₂ -6a), 2.66 (3H, s, CH₃-13), 2.20 (1H, dd, J=8.0 m 2.0 Hz, CH₂ -2a), 2.07 (1H, dd, J=18.0, 6.0Hz, CH₂ -2b), 1.36 (1H, m, CH₂ -15a), 1.24 (1H, m, CH-16), 1.07 (1H, m,CH₂ -15b), 0.80 (3H, d, J=6.3 Hz, CH₃ -17), 0.76 (3H, d, J=6.4 Hz, CH₃-18). delta_(C) (250 MHz, MeOD) 177.13, 173.29, 170.59, 145.02, 133.25,131.38, 119.73, 111.48, 55.51, 42.60, 38.56, 36.68, 26.70, 26.30, 23.46,22.25, 21.05

What is claimed is:
 1. A compound of the formula: ##STR19## wherein R¹is hydrogen, (C₁ -C₆)alkyl, phenyl, phenyl(C₁ -C₆)alkyl;R² is hydrogen,(C₁ -C₆)alkyl, (C₂ -C₆)alkenyl, phenyl (C₁ -C₆)alkyl or cycloalkyl (C₁-C₆)alkyl; R³ represents --CN or a group --O--CH₂ --CO--R⁸ wherein R⁸ isa group --NH--(CH₂)_(n) --COOR¹⁰ where n is 1 or 2, and R¹⁰ is hydrogenor a (C₁ -C₆)alkyl, phenyl or phenyl(C₁ -C₆)alkyl; R⁵ is hydrogen or (C₁-C₆)alkyl; R⁶ is hydrogen or methyl, or salts thereof.
 2. A compound asclaimed in claim 1, in which the chiral center adjacent to thesubstituent R² has R stereochemistry.
 3. A compound as claimed in claim1, in which the chiral center adjacent to the substituted benzyl grouphas S stereochemistry.
 4. A compound as claimed in claim 1, in which thechiral center adjacent to R¹ has S stereochemistry.
 5. A compoundselected from the group consistingof:[4-(N-Hydroxyamino)-2R-isobutylsuccincyl]-L-(4-oxymethylcarboxy-beta-alanine)phenylalanine-N-methylamide;[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxyglycinemethyl ester)phenylalanine-N-methylamide;[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4-oxymethylcarboxy-glycine)phenylalanine-N-methylamide;[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-(4-cyano)phenyl-alanine-N-methylamideand salts thereof.